Quantification of muco-obstructive lung disease variability in mice via laboratory X-ray velocimetry.

To effectively diagnose, monitor and treat respiratory disease clinicians should be able to accurately assess the spatial distribution of airflow across the fine structure of lung. This capability would enable any decline or improvement in health to be located and measured, allowing improved treatment options to be designed. Current lung function assessment methods have many limitations, including the inability to accurately localise the origin of global changes within the lung. However, X-ray velocimetry (XV) has recently been demonstrated to be a sophisticated and non-invasive lung function measurement tool that is able to display the full dynamics of airflow throughout the lung over the natural breathing cycle. In this study we present two developments in XV analysis. Firstly, we show the ability of laboratory-based XV to detect the patchy nature of cystic fibrosis (CF)-like disease in ß-ENaC mice. Secondly, we present a technique for numerical quantification of CF-like disease in mice that can delineate between two major modes of disease symptoms. We propose this analytical model as a simple, easy-to-interpret approach, and one capable of being readily applied to large quantities of data generated in XV imaging. Together these advances show the power of XV for assessing local airflow changes. We propose that XV should be considered as a novel lung function measurement tool for lung therapeutics development in small animal models, for CF and for other muco-obstructive diseases.

Real-time in vivo imaging of regional lung function in a mouse model of cystic fibrosis on a laboratory X-ray source.

Most measures of lung health independently characterise either global lung function or regional lung structure. The ability to measure airflow and lung function regionally would provide a more specific and physiologically focused means by which to assess and track lung disease in both pre-clinical and clinical settings. One approach for achieving regional lung function measurement is via phase contrast X-ray imaging (PCXI), which has been shown to provide highly sensitive, high-resolution images of the lungs and airways in small animals. The detailed images provided by PCXI allow the application of four-dimensional X-ray velocimetry (4DxV) to track lung tissue motion and provide quantitative information on regional lung function. However, until recently synchrotron facilities were required to produce the highly coherent, high-flux X-rays that are required to achieve lung PCXI at a high enough frame rate to capture lung motion. This paper presents the first translation of 4DxV technology from a synchrotron facility into a laboratory setting by using a liquid-metal jet microfocus X-ray source. This source can provide the coherence required for PCXI and enough X-ray flux to image the dynamics of lung tissue motion during the respiratory cycle, which enables production of images compatible with 4DxV analysis. We demonstrate the measurements that can be captured in vivo in live mice using this technique, including regional airflow and tissue expansion. These measurements can inform physiological and biomedical research studies in small animals and assist in the development of new respiratory treatments.

Novel analysis of 4DCT imaging quantifies progressive increases in anatomic dead space during mechanical ventilation in mice.

Increased dead space is an important prognostic marker in early acute respiratory distress syndrome (ARDS) that correlates with mortality. The cause of increased dead space in ARDS has largely been attributed to increased alveolar dead space due to ventilation/perfusion mismatching and shunt. We sought to determine whether anatomic dead space also increases in response to mechanical ventilation. Mice received intratracheal lipopolysaccharide (LPS) or saline and mechanical ventilation (MV). Four-dimensional computed tomography (4DCT) scans were performed at onset of MV and after 5 h of MV. Detailed measurements of airway volumes and lung tidal volumes were performed using image analysis software. The forced oscillation technique was used to obtain measures of airway resistance, tissue damping, and tissue elastance. The ratio of airway volumes to total tidal volume increased significantly in response to 5 h of mechanical ventilation, regardless of LPS exposure, and airways demonstrated significant variation in volumes over the respiratory cycle. These findings were associated with an increase in tissue elastance (decreased lung compliance) but without changes in tidal volumes. Airway volumes increased over time with exposure to mechanical ventilation without a concomitant increase in tidal volumes. These findings suggest that anatomic dead space fraction increases progressively with exposure to positive pressure ventilation and may represent a pathological process.NEW & NOTEWORTHY We demonstrate that anatomic dead space ventilation increases significantly over time in mice in response to mechanical ventilation. The novel functional lung-imaging techniques applied here yield sensitive measures of airway volumes that may have wide applications.

Technical Note: Contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source.

PURPOSE: In vivo imaging of the pulmonary vasculature in small animals is difficult yet highly desirable in order to allow study of the effects of a host of dynamic biological processes such as hypoxic pulmonary vasoconstriction. Here the authors present an approach for the quantification of changes in the vasculature. METHODS: A contrast free angiography technique is validated in silico through the use of computer-generated images and in vivo through microcomputed tomography (µCT) of live mice conducted using a laboratory-based x-ray source. Subsequent image processing on µCT data allowed for the quantification of the caliber of pulmonary vasculature without the need for external contrast agents. These measures were validated by comparing with quantitative contrast microangiography in the same mice. RESULTS: Quantification of arterial diameters from the method proposed in this study is validated against laboratory-based x-ray contrast microangiography. The authors find that there is a high degree of correlation (R = 0.91) between measures from microangiography and their contrast free method. CONCLUSIONS: A technique for quantification of murine pulmonary vasculature without the need for contrast is presented. As such, this technique could be applied for longitudinal studies of animals to study changes to vasculature without the risk of premature death in sensitive mouse models of disease. This approach may also be of value in the clinical setting.

Quantification of heterogeneity in lung disease with image-based pulmonary function testing.

Computed tomography (CT) and spirometry are the mainstays of clinical pulmonary assessment. Spirometry is effort dependent and only provides a single global measure that is insensitive for regional disease, and as such, poor for capturing the early onset of lung disease, especially patchy disease such as cystic fibrosis lung disease. CT sensitively measures change in structure associated with advanced lung disease. However, obstructions in the peripheral airways and early onset of lung stiffening are often difficult to detect. Furthermore, CT imaging poses a radiation risk, particularly for young children, and dose reduction tends to result in reduced resolution. Here, we apply a series of lung tissue motion analyses, to achieve regional pulmonary function assessment in ß-ENaC-overexpressing mice, a well-established model of lung disease. The expiratory time constants of regional airflows in the segmented airway tree were quantified as a measure of regional lung function. Our results showed marked heterogeneous lung function in ß-ENaC-Tg mice compared to wild-type littermate controls; identified locations of airway obstruction, and quantified regions of bimodal airway resistance demonstrating lung compensation. These results demonstrate the applicability of regional lung function derived from lung motion as an effective alternative respiratory diagnostic tool.

Imaging lung tissue oscillations using high-speed X-ray velocimetry.

This work utilized synchrotron imaging to achieve a regional assessment of the lung's response to imparted oscillations. The forced oscillation technique is increasingly being used in clinical and research settings for the measurement of lung function. During the forced oscillation technique, pressure oscillations are imparted to the lungs via the subjects' airway opening and the response is measured. This provides information about the mechanical properties of the airways and lung tissue. The quality of measurements is dependent upon the input signal penetrating uniformly throughout the lung. However, the penetration of these signals is not well understood. The development and use of a novel image-processing technique in conjunction with synchrotron-based imaging was able to regionally assess the lungs' response to input pressure oscillation signals in anaesthetized mice. The imaging-based technique was able to quantify both the power and distribution of lung tissue oscillations during forced oscillations of the lungs. It was observed that under forced oscillations the apices had limited lung tissue expansion relative to the base. This technique could be used to optimize input signals used for the forced oscillation technique or potentially as a diagnostic tool itself.

Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo.

Every cell in our body originates from the pluripotent inner mass of the embryo, yet it is unknown how biomechanical forces allocate inner cells in vivo. Here we discover subcellular heterogeneities in tensile forces, generated by actomyosin cortical networks, which drive apical constriction to position the first inner cells of living mouse embryos. Myosin II accumulates specifically around constricting cells, and its disruption dysregulates constriction and cell fate. Laser ablations of actomyosin networks reveal that constricting cells have higher cortical tension, generate tension anisotropies and morphological changes in adjacent regions of neighboring cells, and require their neighbors to coordinate their own changes in shape. Thus, tensile forces determine the first spatial segregation of cells during mammalian development. We propose that, unlike more cohesive tissues, the early embryo dissipates tensile forces required by constricting cells via their neighbors, thereby allowing confined cell repositioning without jeopardizing global architecture.

In vivo wall shear measurements within the developing zebrafish heart.

Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.

Optimisation of a stirred bioreactor through the use of a novel holographic correlation velocimetry flow measurement technique.

We describe a method for measuring three dimensional (3D) velocity fields of a fluid at high speed, by combining a correlation-based approach with in-line holography. While this method utilizes tracer particles contained within the flow, our method does not require the holographic reconstruction of 3D images. The direct flow reconstruction approach developed here allows for measurements at seeding densities in excess of the allowable levels for techniques based on image or particle reconstruction, thus making it suited for biological flow measurement, such as the flow in bioreactor. We outline the theory behind our method, which we term Holographic Correlation Velocimetry (HCV), and subsequently apply it to both synthetic and laboratory data. Moreover, because the system is based on in-line holography, it is very efficient with regard to the use of light, as it does not rely on side scattering. This efficiency could be utilized to create a very high quality system at a modest cost. Alternatively, this efficiency makes the system appropriate for high-speed flows and low exposure times, which is essential for imaging dynamic systems.